Small cell lung cancer (SCLC) is an understudied cancer for which no molecularly targeted approaches have shown use. Here we propose to use a chemical biology platform, activity-based protein profiling (ABPP), to study the small cell lung cancer (SCLC) proteome. ABPP uses chemical probes that are directed against the active sites of enzymes to interrogate the functional state of enzymes in biological samples. Using ABPP, a number of enzyme families have been studied, including serine hydrolases, kinases, phosphatases, and metalloproteinases. We propose to use ABPP to better classify and subgroup SCLC based on global protein kinase profiling that can guide future attempts towards developing targeted therapy. The hypothesis for this project is that kinome-based ABPP can detect functional kinases in SCLC and guide future clinical management by (i) identifying active kinases and pathways that are potential drivers of SCLC growth and survival, (ii) can lead to development of biomarkers that classify different subgroups of SCLC, and (iii) can inform about combination therapy approaches that can be further studied for effectiveness in SCLC. Proteomic profiling offers additional views of SCLC biology beyond sequencing of genes or gene expression profiles and thus is complementary to other important work examining SCLC biology. To enable the project goals, we have proposed four aims. Aim 1 will develop quantitative methods to characterize kinome and other ATP-binding proteins using mass spectrometry. Aim 2 will characterize kinases and other ATP-binding proteins in SCLC tumors and cell lines using ABPP. Aim 3 will validate findings of ABPP findings using western blotting and Reverse Phase Protein Arrays. Aim 4 will interrogate the functional significance of ABPP identified proteins kinases using RNAi and small molecule inhibitors to better guide biomarker selection.